Effect of Expression of Human Spermidine/Spermine Nl -Acetyltransferase in Escherichia coli

Lisa Parry, Juan Lopez-Ballester, Laurie Wiest, Anthony E. Pegg

Research output: Contribution to journalArticlepeer-review

21 Scopus citations

Abstract

A plasmid expression vector, pINSAT2, was constructed in order to express spermidine/spermine N1-acetyltransferase (SSAT) in Escherichia coli. Cells transfected with this vector produced large amounts of SSAT, amounting to up to 2% of the soluble protein when isopropyl β-D-thiogalactopyranoside (IPTG) was added and 0.3% of the soluble protein in the absence of inducer. The growth rate of cells expressing SSAT was reduced, and all of the cellular spermidine was converted to (N1-acetylspermidine, much of which was excreted. Putrescine and 1 -methylspermidine, which is not a substrate for SSAT, could reverse the effects of SSAT expression on growth, but spermidine was only effective when the amount of SSAT expression was limited by omitting the IPTG inducer. The lack of stimulation of growth by spermidine correlated with its complete conversion to N1-acetylspermidine. These results show that N1-acetylspermine is not able to substitute for the unmodified polyamines in supporting growth and suggest that acetylation is a physiological response to convert excess polyamines to a physiologically inert form which is readily excreted. Cells expressing large amounts of SSAT were much more senstive to the growth inhibitory action of the antitumor agent Nl, N12-bis(ethyl)spermine, supporting the hypothesis that the ability of such bis(ethyl) polyamines to induce SSAT contributes to their antiproliferative actions. SSAT was readily purified to homogeneity from extracts of DH5α cells containing pINSAT2. The purified enzyme had a simialr specific activity and Km values for spermine and spermidine as the enzyme purified from human colon cancer cells, suggesting that posttranslational modifications specific to eukaryotes are not needed for enzymatic activity. The recombinant SSAT was found to acetylate the drugs 15-deoxyspergualin, 2-[(aminopropyl)amino]ethanethiol, and N-(n-butyl)-1,3-diaminopropane.

Original languageEnglish (US)
Pages (from-to)2701-2709
Number of pages9
JournalBiochemistry
Volume34
Issue number8
DOIs
StatePublished - Feb 1995

All Science Journal Classification (ASJC) codes

  • Biochemistry

Fingerprint Dive into the research topics of 'Effect of Expression of Human Spermidine/Spermine N<sup>l</sup> -Acetyltransferase in Escherichia coli'. Together they form a unique fingerprint.

Cite this